Expression and secretion vector in yeasts, useful for preparing heterologous proteins

Abstract

The invention provides an expression and secretion vector in yeasts which is useful for preparing heterologous proteins, comprising a synthetic oligonucleotide which directs the secretion of the heterologous protein wherein the synthetic oligonucleotide is positioned between the inducible hybrid promoter GAL-CYC and a multiple-site polylinker followed by the signals of transcription termination recognized by the RNA polymerase of the yeasts. The invention includes a hybrid plasmid obtained by cloning in one of the restriction sites of the polylinker of the vector, the DNA sequence which codes for a heterologous protein. A process is also described for the preparation of heterologous proteins which comprises cultivating in a suitable culture medium a yeast transformed with the hybrid plasmid and recovering from the culture medium the resulting heterologous proteins. The recovered proteins include hormones, lymphokines, viral antigens or immunogens useful in the therapeutical and diagnostic field.

We claim: 
     
       1. A cloning vector for expression and secretion in yeasts, containing the inducible promoter GAL-CYC, the FLP termination signal sequence of transcription recognized by yeasts and one secretion signal sequence consisting of: ##STR3## 
     
     
       2. The cloning vector of claim 1, wherein the secretion signal sequence encodes a 19-amino acid peptide having the following amino acid sequence: Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Gln Gly Thr Arg Gly. 
     
     
       3. The cloning vector of claims 1 or 2 obtained by a process consisting of the steps of: (a) building the expression vector pEMBLyex2 containing the leu2-d gene, the replication origin of the F1 phage, the gene coding for resistance to ampicillin, the gene uracil-3, the inducible hybrid promoter GAL-CYC derived for G2 and the polylinker of pEMBL18;   (b) digesting the pEMBLyex2 with the restriction enzymes SstI and KpnI which cut in the restriction siste of the polylinker;   (c) inserting in the restriction sites SstI and KpnI of the plasmid obtained in stage (b) the synthetic nucleotide which has the following sequence: ##STR4##  (d) isolating the resulting expresion and secretion vector.   
     
     
       4. A cloning vector for expression and secretion in yeasts of interleulin-1β, containing the inducible promoter GAL-CYC, the FLP termination signal sequence of transcription recognized by yeast and one secretion signal sequence consisting of: ##STR5## 
     
     
       5. S. Cerevisiae containing a hybrid plasmid that expresses and secretes human interleukin 1β, said hybrid plasmid comprising a heterologous gene coding for said human interleukin 1β and being under the control of the promoter, secretion signal sequence, and termination sequence contained in the cloning vector according to claims 1 or 2. 
     
     
       6. An E. coli cell which secretes human interleukin 1β, said cell containing the hybrid plasmid YEpsec1-hI-1β, deposited as ATCC number 67024. 
     
     
       7. A hybrid plasmid contained in a usable form of S. cerevisiae to express and secrete human interleukin 1β which is YEpsec1-hI-1β.